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Abstract Mt LPMO9 and Fo LPMO9 are two lytic polysaccharide monooxygenases (LPMOs), from the filamentous fungi Thermothelomyces thermophila and Fusarium oxysporum , respectively. In the present study an attempt has been made to achieve direct electron transfer between these enzymes and a glassy carbon electrode by immobilization in Nafion polyelectrolyte. The method used to ascertain the feasibility of direct electron transfer was large amplitude Fourier transform alternating current voltammetry (FTacV) and the formal potentials of these enzymes were determined at different temperatures. The findings of this paper indicate that LPMOs can be studied by direct electron transfer, which could be exploited in the near future for their biochemical characterization. Highlights • Direct electron transfer is detected for Mt LPMO9 and Fo LPMO9 by FTacV. • The formal potentials are estimated by FTacV. • The temperature dependence of formal potentials is determined. [ABSTRACT FROM AUTHOR]

• Kinetic analysis of an immobilized enzyme/free substrate reaction for FTacV is made. • The dependence of FTacV chief observables on substrate concentration is analyzed. • The direct electron transfer of a multicopper oxidase is ascertained. • The detection of epinephrine by immobilized multicopper oxidase is achieved. In the present work, a kinetic analysis is made concerning the reaction of an electroactive immobilized enzyme with a free substrate, based on a Michaelis–Menten scheme. The proposed kinetic equations are investigated numerically for conditions describing large amplitude fast Fourier transform alternating current voltammetry (FTacV), under different reaction states (transient or steady state for the reaction intermediate as well as quasi or complete reversibility of the electrochemical step). The dependence of two chief observables that occur from the analysis of the results of the method, that is, the maximum of the harmonics and the potential shift of the corresponding dominant peaks, on substrate concentration is presented. The FTacV method is applied experimentally for an immobilized laccase-like multicopper oxidase from Thermothelomyces thermophila , Tt LMCO1, and its reaction with epinephrine. From the experimental findings it is shown that the intrinsic characteristics of the system do not lead to the extraction of the desired kinetic data although indications on the relation between the kinetic constants is revealed. Finally, the response of the third harmonic for the first additions of epinephrine at subnanomolarity range can be exploited for the detection of epinephrine at rather low concentrations. [ABSTRACT FROM AUTHOR]